Tetsuo Shiota

The oxidative cleavage of folates: A critical study

Abstract Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C9N10 bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N5-formyl-tetrahydrofolic acid are cleaved at the C9N10 bond to produce p-aminobenzoylglutamic acid. N5, N10-methenyl-tetrahydrofolic acid, N5,N10-methylene-tetrahydrofolic acid, and N10-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N10-formyl-folic acid which is completely stable to the oxidative treatment employed. N5-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N5-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5-3H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-14C]folic acid to label the folates in vivo is presented.

A differential microdetermination for the various forms of biopterin.

Abstract Conditions for the quantitative oxidation and destruction of tetrahydrobiopterin and quinoid dihydrobiopterin and the separation of biopterin from its reduced forms by ECTEOLA-Sephadex column chromatography are described. A procedure for the quantitation of tetrahydrobiopterin plus quinoid dihydrobiopterin, 7,8-dihydrobiopterin, and biopterin using a Crithidia bioassay is presented. Using these procedures it was found that tetrahydrobiopterin plus quinoid dihydrobiopterin are the prevalent forms in liver and blood of mice and that biopterin was the predominant form in the tails of tadpoles. In human urine, approximately half of the biopterin was found as tetrahydrobiopterin plus quinoid dihydrobiopterin and the other half was 7,8-dihydrobiopterin. The presence of tetrahydrobiopterin and quinoid dihydrobiopterin was confirmed by a coenzyme assay for the hydroxylation of phenylalanine.

Virulence of Streptococcus mutans: Biochemical and Pathogenic Characteristics of Mutant Isolates

Summary The in vitro dextran-sucrase activities and adherence to glass of S. mutans 6715 and PS14 wild types and mutants were quantitated and compared with their in vivo cariogenicity in young, gnotobiotic rats. In general, S. mutans PS14 mutants B414 and B421 and 6715 mutant C4 demonstrated less dextran-sucrase activity and adherence than parental strains and caused fewer carious lesions in gnotobiotic rats. Rats monoinfected with either PS14 mutants B414 or B421 had less plaque and viable S. mutans in plaque than rats infected with parental strain. Both S. mutans 6715 mutants C211 and C229, demonstrated greater enzyme activity and adherence than the parental strain and produced more carious lesions.

The preparation of a modified GTP-sepharose derivative and its use in the purification of dihydroneopterin triphosphate synthetase, the first enzyme in folate biosynthesis

Abstract The conditions for coupling periodate oxidized GTP to a hydrazide Sepharose derivative are described. Approximately 1 μmole of the ligand was bound per milliliter of settled gel. Gel columns prepared from this material bind D- erythrod ihydroneopterin triphosphate synthetase, the initial enzyme for folate biosynthesis in Lactobacillus plantarum . A yield of 28% and an overall enzyme purification of 765 fold were attained when the affinity technique was used with a conventional purification procedure.

The assignment of structure to the formamidopyrimidine nucleoside triphosphate precursor of pteridines

Abstract An open-ring compound, Compound V, which was prepared chemically from GTP was previously found to be a pteridine precursor using extracts of Lactobacillus plantarum . The structure of Compound V was established to be 2-amino-6(5′-triphosphoribosyl) am amino-5- or -6-formamido-4-hydroxypyrimidine. By a procedure involving hydrolysis in 90–98% formic acid and chromatography, the hydrolyzed product was found to have properties identical to 2,6-diamino-5-formamido-4-hydroxypyrimidine. These results indicate that Compound V is 2-amino-6(5′-triphosphoribosyl ) amino-5-formamido-4-hydroxypyrimidine.

The enzymatic synthesis of Crithidia active substance(s) and a phosphorylated D-Erythroneopterin from GTP or GDP by liver preparations from syrian golden hamsters

Summary Dialyzed liver homogenates from the Syrian golden hamster were found to be enzymatically active for the production of Crithidia active substance(s) from GTP or GDP. This activity was stimulated by a mixture of pyridine nucleotides. An Ultrogel AcA-34 fraction of a liver homogenate preparation produced a fluorescent substance from GTP which was not biopterin. This fluorescent substance was identified as a phosphorylated derivative of D- erythro neopterin.

Taxonomy of the oral Streptococcus mutans based on colonial characteristics and serological, biochemical and genetic features

Abstract Strains of Streptococcus mutans representing 6 different serotypes were cultured in pour plates containing a glucose agar medium under an atmosphere of 95 per cent nitrogen-5 per cent carbon dioxide. After growth, a sucrose solution was added to these plates. Three colonial types were obtained: type 1 colonies were rough spheroid shaped, with no white haze around any colony; type 2 were rough spheroid shaped with a white haze around each colony; type 3 were smooth lens shaped with white haze. On the other hand, all strains tested grew as rough spheroid shaped colonies in a sucrose agar medium pour plates. When these groupings were compared with groupings based on serological, genetic and biotype characterization demonstrated that strains belonging to the colonial type 1 were of serotype c or e , genetic group I and biotype I or V. Strains belonging to the colonial type 3 were of serotype a , d , or g , genetic group III or IV.

The nature of the multiple forms of d-Erythro-dihydroneopterin triphosphate synthetase

1. Three forms of the Lactobacillus plantarum enzyme D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme in folate biosynthesis, have been demonstrated by polyacrylamide gel electrophoresis. The enzyme forms designated the alpha prime, alpha and beta forms have been shown to be conformers with molecular weights of approx. 200 000. Study of the subunit structure of the beta enzyme species by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed a single protein with an estimated molecular weight of 20 000 which suggests that the enzyme molecule may be composed of ten polypeptide chains. 2. Of the three conformers only one form, the beta form, appears to be enzymatically active. The two other conformers must undergo conformational changes to the beta species before enzymatic activity can be demonstrated in reaction mixtures containing these enzyme forms. 3. The three enzyme species are interconvertible. The removal of phosphate ions from the enzymatically active beta form results in the formation of two inactive species which suggests that the conformation of the active enzyme is stabilized by non-covalently bound phosphate ions. Conversion of the inactive species to the beta enzyme form may be effected by the readdition of phosphate, substrate or certain nucleotides.

An improved method for measuring aggregation of certain streptococcal bacteria found in dental plaque

Various cells were incubated with either dextran or sucrose and the reaction was terminated by the addition of a 20 per cent solution of formaldehyde. A reaction mixture consisting of cells and aggregated cells was applied to a step-gradient glycerol column (0-60 per cent) and 0.5 ml fractions were collected from the bottom of the column. Non-aggregated cells remained in the top layer of the column. Aggregated cells settled in the 30 per cent glycerol layer as determined by either spectrophotometric or radioactive methods. The amount of aggregated cells demonstrated in this layer increased with either incubation time or concentration of sucrose, dextran or cells. Cells of Strep, mutans strains 6715, AHT, 10449, OMZ-176, OMZ-175 and two mutants of Strep. mutans strain 6715 (designated C4 and C307) aggregated strongly. Cells of Strep. mutans strain LM7 aggregated weakly, whereas cells of Strep. mutans 6715 mutant UAB165, a mutant defective in aggregation. Strep. mutans strain BHT, Strep. salivarius and Strep. sanguis did not aggregate in the presence of either sucrose or dextran. Experiments testing co-aggregation between two different types of cells were done by using 3H-labelled and 14C-labelled cells. Strep. mutans strain 6715 co-aggregated with Strep. sanguis in the presence of sucrose or dextran, whereas Strep. mutans strain 6715 did not aggregate with Strep. salivarius. The method separates aggregated from non-aggregated cells and enables quantitation of sucrose- or dextran-induced aggregation and co-aggregation.

Use of Mutants in the Elucidation of Virulence of Streptococcus Mutans

In the presence of sucrose, Streptococcus mutans displays certain unique metabolic activities which are believed to be important factors in determining its virulence. Of the various carbohydrates metabolized by S. mutans, only sucrose is utilized for the production of acid, fructans and glucans (1,2). The adhesive water-insoluble glucans have been shown to be most important for the attachment of cells of S. mutans to smooth surfaces (1,3,5); probably both the water-insoluble and water-soluble glucans are required for the cell to cell aggregation which results in plaque production (6,7). Several laboratories have employed bacterial mutants and model systems of cell adherence and aggregation to study the cariogenic nature of S. mutans (5,8–13). We present results of certain biochemical activities of mutants of S. mutans strain 6715 and attempt to relate these findings to the cariogenic and immunogenic nature of these organisms.